Compositions and methods for treating joints

ABSTRACT

Compositions and methods are provided for treating joint conditions, such as osteoarthritis and/or the pain associated therewith. The compositions and methods utilize a first component, namely hyaluraonic acid (“HA”), in combination with a lyophilized second component that is effective to at least temporarily reduce the viscosity of the HA. In an exemplary embodiment, the second component is one or more glycosaminoglycans (“GAG”), such as chondroitin sulfate (“CS”), including CS4 and/or CS6, dermatan sulfate, heparin, heparan sulfate, and keratan sulfate. The composition can optionally include other joint supplements, such as glucosamine (“GlcN”)

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/750,148 filed on Jan. 25, 2013 and entitled “Compositions and Methodsfor Treating Joints,” which is a continuation of U.S. patent applicationSer. No. 12/979,981 filed on Dec. 28, 2010 and entitled “Compositionsand Methods for Treating Joints,” and which issued as U.S. Pat. No.8,398,611 on Mar. 19, 2013, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fortreating joints.

BACKGROUND OF THE INVENTION

Osteoarthritis (“OA”), the most common form of arthritis, is a type ofarthritis that is characterized by degenerative (gradual deteriorationof joint) or abnormal changes in bone, cartilage, and synovium of thejoints. OA is often characterized by a progressive wearing down ofopposing joint surfaces accompanied at times by inflammation resultingin pain, swelling, and stiffness for the patient . OA can occur in oneor more joints following trauma to the joint, following an infection ofthe joint, or simply as a result of aging. Furthermore, there isemerging evidence that abnormal anatomy may contribute to earlydevelopment of OA.

Treatment of OA generally involves a combination of exercise or physicaltherapy, lifestyle modification, and analgesics. Acetaminophen istypically the first line treatment for OA. For mild to moderatesymptoms, effectiveness is similar to non-steroidal anti-inflammatorydrugs (“NSAIDs”), such as ibuprofen. For more severe symptoms NSAIDs maybe more effective. However, while more effective, NSAIDs in severe casesare associated with greater side effects such as gastrointestinalbleeding and renal complications. Another class of NSAIDs, COX-2selective inhibitors (such as Celecoxib), are equally effective toNSAIDs but no safer in terms of side effects. There are several NSAIDsavailable for topical use including diclofenac. Typically, they haveless systemic side-effects than oral administration and at least sometherapeutic effect. While opioid analgesic, such as morphine andfentanyl, improve pain this benefit is outweighed by frequent adverseevents and thus they are not routinely used. Intra-articular steroidinjections are also used in the treatment of OA and they are veryeffective at providing pain relief. However, the durability of the painrelief is limited to 4-6 weeks and there are adverse effects that mayinclude collateral cartilage damage. If pain becomes debilitating, jointreplacement surgery may be used to improve mobility and quality of life.There is no proven treatment to slow or reverse the disease.

For patients who do not get adequate pain relief from simple painrelievers, like acetaminophen or from exercise and physical therapy,intra-articular injections of hyaluronic acid (HA) provide anothertreatment option to address symptomatic pain and delay the need for atotal joint replacement surgery. It is known that the concentration ofnative HA is deficient in individuals suffering from OA and thereforejoint injections of exogenous HA is believed to replenish thesemolecules and restore the viscoelastic properties of synovial fluid. Itis this property that is responsible for lubricating and cushioning thejoints. There is also evidence that HA has biological activity throughbinding to cell surface receptors and may have a role in mitigatinginflammation. Independent of the mechanism of action, pain relief isobserved for about six months following a treatment course. A treatmentcourse for HA products on the US market can range from single injectionproduct to others that require 3 to 5 weekly injections to attain thisdurability of pain relief.

There remains a need for improved methods and compositions for treatingOA joints, and in particular to improved methods and compositions fortreating joints using HA combined with one or more GAGs to address thepain and structural degeneration associated with OA.

SUMMARY OF THE INVENTION

The present invention generally provides compositions and methods fortreating joint conditions, such as osteoarthritis and/or the painassociated therewith. In one embodiment, a kit for treating joints isprovided and includes a first component comprising hyaluronic acid(“HA”), a second component comprising a lyophilized glycosaminoglycan(“GAG”), and a syringe for injecting a mixture of the first and secondcomponents. While various GAGs can be used, in one embodiment the GAGcomprises chondroitin sulfate. The kit can also include a thirdcomponent, such as glucosamine, which in some embodiments can belyophilized.

The composition of the components can vary. In one embodiment, when thefirst and second components are combined to form a mixture, the firstand second components are present within the mixture at a ratio of firstcomponent to second component in the range of about 1:0.005 to 1:100 byweight. In other aspects, the hyaluronic acid can be lyophilized. In oneexemplary embodiment, the hyaluronic acid has a molecular weight in therange of about 1 million Daltons (MDa) to 4 MDa, and a concentration ofat least about 5 mg/ml, and more preferably at least about 7 mg/ml. Thekit can also include a third component comprising a fluid that iseffective to solubilize the first and second components. The fluid canbe, for example, water, saline, and/or a buffer.

The syringe can have various configurations, and in one embodiment thesyringe has a first chamber containing the first component, a secondcontainer containing the second component, and a plunger configured toinject the second component into the first chamber to mix the first andsecond components. In another embodiment, the second component can bedisposed within a container, and the syringe can include a first chambercontaining the first component and a connector for removably couplingthe container to the syringe such that a plunger slidably disposedwithin the first chamber of the syringe can inject the first componentinto the container to mix the first and second components.

In other aspects, a method for treating joints is provided and includescombining a first component comprising hyaluronic acid with a secondcomponent comprising a lyophilized glycosaminoglycan to form a mixture,and injecting the mixture into a joint. The glycosaminoglycan can be,for example, chondroitin sulfate. The kit can also include a thirdcomponent, such as glucosamine. In certain aspects, the glucosamine, ifpresent, can be lyophilized. When the components are combined, aviscosity of the mixture is less than a viscosity of the hyaluronic acidprior to combining. In another embodiment, the hyaluronic acid islyophilized, and the first and second components are solubilized priorto or during combining the first and second components to form amixture. The first and second components can be combined, for example,in a vial and drawn into a syringe, which can be used to inject themixture into a joint. In an exemplary embodiment, the first and secondcomponents are drawn into the syringe after forming the mixture, forexample within about 30 minutes of formation of the mixture. In anotherembodiment, the first component is disposed within a first chamber in asyringe, and the second component is disposed within a second chamber inthe syringe, and combining the first and second components comprisesinjecting the first component into the second chamber. In yet anotherembodiment, combining the first and second components comprises couplinga container having the first component disposed therein to a syringehaving the second component disposed therein, and injecting the secondcomponent from the syringe into the container.

The first component can have various concentrations and molecularweights, but in one embodiment the concentration is at least about 5mg/ml, and more preferably at least about 7 mg/ml and the molecularweight is in the range of about 1 MDa to 4 MDa. In an exemplaryembodiment, the first and second components are present within themixture at a ratio of about 1:0.005 to 1:100.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects will become clear and canbe understood in detail. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate exemplary embodiments and should not be considered to limitthe scope.

FIG. 1 is a perspective view of one embodiment of a mixing and deliverysystem for use with the compositions and methods of the presentinvention; and

FIG. 2 is a perspective view of another embodiment of a mixing anddelivery system for use with the compositions and methods of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention provides compositions and methods fortreating joint conditions, such as osteoarthritis and/or the painassociated therewith. The compositions and methods utilize a firstcomponent, namely hyaluronic acid (“HA”), in combination with alyophilized second component that is effective to at least temporarilyreduce the viscosity of the HA. In an exemplary embodiment, the secondcomponent is one or more glycosaminoglycans (“GAG”), such as chondroitinsulfate (“CS”), including CS4 and/or CS6, dermatan sulfate, heparin,heparan sulfate, and keratan sulfate. The composition can optionallyinclude other joint supplements, such as glucosamine (“GlcN”).

It has been discovered that the use of a lyophilized GAG has the effectof at least temporarily reducing the viscosity of HA such that acomposition formed from lyophilized GAG and HA has a viscosity that is(at least temporarily) significantly less than the viscosity of the HAalone. While it is desirable to use HA at high molecular weights andhigh concentrations, the viscosity of HA significantly increases as themolecular weight and concentration increases. As a result, HA istypically either used at low molecular weights and concentrations toallow for intra-operative mixing, or HA is mixed during manufacture.Mixing the HA in advance of the time of its intended use, such as at thetime of manufacture, can present issues with respect to the stabilityand shelf life of the HA composition. The discovery that lyophilized GAGis effective to reduce the viscosity of the HA, at least temporarily,enables the use of HA at high molecular weights and concentrations, thusallowing it to be mixed with GAG intra-operatively, i.e., immediatelyprior to or during surgery, and thereby eliminating any compositionstability and shelf life issues. This decrease in viscosity issufficient to allow mixing to take place without the use of anymechanical aid, such as a vortexer, mixer, or a spatula. Therefore,there is need for specific instruments or stirrers that could compromisesterility to create a formulation that combines HA and CS.

First Component

The first component, HA, can have various formulations and can beprovided at various concentrations and molecular weights. The terms“hyaluronic acid,” “hyaluronan,” and “HA” are used interchangeablyherein to refer to hyaluronic acids or salts of hyaluronic acid, such asthe sodium, potassium, magnesium, and calcium salts, among others. Theseterms are also intended to include not only elemental hyaluronic acid,but hyaluronic acid with other trace elements or in various compositionswith other elements. The terms “hyaluronic acid,” “hyaluronan,” and “HA”encompass chemical or polymeric or cross-linked derivatives of HA.Examples of chemical modifications which may be made to HA include anyreaction of an agent with the four reactive groups of HA, namely theacetamido, carboxyl, hydroxyl, and the reducing end. The HA used in thepresent application is intended to include natural formulations,synthetic formulations, or combinations thereof. The HA can be providedin liquid or solid formulations, and the HA can be in pure liquid formor in a solvent at various concentrations.

HA is a glycosaminoglycan (GAG), and in particular HA is an unbranchedpolysaccharide made up of alternating glucuronic acid and N-acetylglucosamine units. It is a viscoelastic material that is also found inthe extracellular matrix of cartilage attached to collagen. Inparticular, HA is an important building component of aggregatedproteoglycans which impart resilient characteristics of articularcartilage. HA not only helps keep the cartilage that cushions jointsstrong and flexible, but it also helps increase supplies ofjoint-lubricating synovial fluid. HA abnormalities are a common threadin connective tissue disorders. HA can thus be used, to prevent, treat,or aid in the surgical repair of connective tissue disorders.

HA can be used in the compositions and methods of the present inventionat various molecular weights. Since HA is a polymeric molecule, the HAcomponent can exhibit a range of molecular weights, and almost anyaverage of modal molecular weight formulation of HA can be used in thecompositions and methods of the present invention, including LowMolecular Weight (“LWM”) Hyaluronan (about 500 to 700 kilodaltons (kDa)Medium Molecular Weight (“MMW”) Hyaluronan (700-1000 kDa), and HighMolecular Weight (“HMW”) Hyaluronan (1.0-4.0 million daltons (MDa)). Incertain exemplary embodiments, the HA has a molecular weight of at leastabout 500 kDa, and more preferably the HA is a High Molecular Weight(“HWM”) HA having a molecular weight of at least about 1 MDa. Themolecular weight can be, for example, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400,3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600,4700, 4800, 4900, 5000 kDa or more, or any range derivable therein. Itis expected that chemically modified HA's could have very differentmolecular weights than described above. A crosslinked HA can have muchhigher molecular weight than noted above. Regardless, these materialsare also applicable in this invention preferably when combined with anon-crosslinked or lightly crosslinked HA in the formulation.

The concentration of HA present in mixture can also vary, but in anexemplary embodiment HA is provided at a pharmaceutically effectiveamount. In an exemplary embodiment, the HA has a concentration of atleast about 5 mg/ml, and more preferably at least about 7 mg/ml, andmore preferably at least about 10 mg/ml, and more preferably at leastabout 15 mg/ml, and in some embodiments the concentration can be atleast about 20 mg/ml. Suitable concentrations of HA include about 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/mg, 12 mg/ml,13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59mg/ml, 60 mg/ml or more or anyrange derivable therein.

In one embodiment, the first component comprises an HA having a highmolecular weight (e.g., 1 to 4 MDa) and a concentration in the range ofabout 7-40 mg/ml. One such product is Orthovisc® manufactured by AnikaTherapeutics, Inc. of Bedford, Mass. Orthovisc® is a sterile,non-pyrogenic, clear, viscoelastic solution of hyaluronan. Orthovisc®consists of high molecular weight (1.0-2.9 MDa), ultra-pure naturalhyaluronan dissolved in physiological saline and having a nominalconcentration of 15 mg/ml. Orthovisc® is isolated through bacterialfermentation. A person skilled in the art will appreciate that HMW HAcan be obtained from a number of sources, such as Shiseido and Lifecore.Another example of an HA product in the US with these characteristics isEuflexxa®.

Second Component

The second component used in the methods and compositions of the presentinvention can also have a variety of configurations, but in an exemplaryembodiment the second component is a lyophilized glycosaminoglycan(GAG). The term “glycosaminoglycan,” or “GAG,” refers interchangeably tothe family of sulfated mucopolysaccharides that typically includeheparin, heparin sulfate, chondroitin, chondroitin sulfate, keratansulfate, dermatan sulfate, and their respective derivatives.

Glycosaminoglycans (GAGs) are long unbranched polysaccharides containinga repeating disaccharide unit. The disaccharide units contain either oftwo modified sugars, N-acetylgalactosamine (GalNAc) orN-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate oriduronate. GAGs are highly negatively charged molecules, with extendedconformation that imparts high viscosity to the mixture. GAGs arelocated primarily on the surface of cells or in the extracellular matrix(ECM). Along with the high viscosity of GAGs comes low compressibility,which makes these molecules ideal for a lubricating fluid in the joints.GAGs can thus help slow down the inflammatory process. At the same time,their rigidity provides structural integrity to cells and providespassageways between cells, allowing for cell migration. In addition, aGAG such as CS has been found in the synovial fluid and may play a rolein health of the joint. Therefore it makes sense to have a therapy thatcan deliver CS at the same time as HA. It is believed that CS canenhance the efficacy of HA. However, we have observed that HA in thepresence of CS can degrade over time. Therefore, in order to preventthis degradation from taking place, it needs to be stored refrigerateduntil use. However, we know that most clinics and hospital do not havethe capacity to store these types of products in a refrigeratortherefore we needed to find a way to provide products that can combineHA and GAG while maintaining the ability to keep them at roomtemperature.

One way to increase stability of a formulation is to provide the productas a lyophilized product that can be resuspended with a diluent justprior to or at the time of injection. For example, a lyophilized GAG canbe provided for resuspending with an HA gel just prior to or at the timeof injection. Unfortunately, HA's at high molecular weights and/or highconcentrations are extremely viscous. Even with this high viscosity,surgeons are able to deliver the formulation into the joint because HA,especially uncrosslinked HA's, have lower viscosity with increasingshear rate. This phenomenon, called shear thinning, occurs when the HAis pushed through a needle. The HA shear thins or the viscositydecreases which helps to facilitate the injection into that patient'sjoint. When the HA is not under a mechanical force, the viscosityremains high and makes it difficult to be able to dissolve and mixanother active into the HA without any mechanical aid or mixing system.

It has been discovered that when HA is combined with GAGs, and inparticular lyophilized GAGs, the GAG, once dissolved, can at leasttemporarily reduce the viscosity of HA sufficiently to allow mixing totake place. The advantage of a lyophilized GAG formulation over a liquidGAG formulation is that it allows the HA component to be used at higherconcentrations. While a liquid GAG can be combined with HA, such amixture of liquid GAG and HA would yield a lower concentration of HA. Itis believed that higher concentrations of HA's are desirable to providedurability of pain relief in the clinical setting. While various GAGscan be used, in certain exemplary embodiments the lyophilized GAG issulfated, and more particularly it is one or more of chondroitin sulfate(CS), including CS4 and/or CS6, dermatan sulfate, heparin, heparansulfate, and keratan sulfate.

The GAGs can have various molecular weights, but in certain exemplaryembodiments the molecular weight is in the range about 5 to 1,000 kDa,more preferably in the range of about 6 to 500 kDa, more preferably inthe range of about 7 to 300 kDa, more preferably in the range of about 8to 200 kDa, more preferably in the range of about 9 to 100 kDa, and mostpreferably in the range of about 10 to 80 kDa. In other embodiments, themolecular weight of the GAG fragment is below about 5 kDa and even morepreferably below about 3 kDa.

The concentration of GAGs present in mixture can also vary, but in anexemplary embodiment the GAG is provided at a pharmaceutically effectiveamount. In an exemplary embodiment, the GAG has a concentration of atleast about 0.1 mg/ml, and more preferably at least about 2 mg/ml, andmore preferably at least about 5 mg/ml, and more preferably at leastabout 7 mg/ml. Suitable concentrations of GAGs include about 5 mg/ml, 6mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/mg, 12 mg/ml, 13mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml, 60 mg/ml or more or anyrange derivable therein.

Chondroitin sulfate (CS), which is an essential component of cartilage,is composed of an alternating sequence of sulfated and/or unsulfatedD-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (Ga1NAc) residueslinked through alternating β(1,3) and β(1,4) bonds. These compounds eachhave a polymeric structure consisting mainly of about 40 to 100 timesrepetition of the disaccharide units. CS can be used at variousmolecular weights and concentrations, as discussed above with respect tothe GAG component, but in an exemplary embodiment, the CS has amolecular weight of in the range of about 10,000 to 80,000 kDa and aconcentration in the range of about 0.1 to 100 mg/ml. CS can be isolatedfrom bovine or marine sources. A chondroitin chain can have over 100individual sugars, each of which can be sulfated in variable positionsand quantities. Chondroitin-4 sulfate, also carbon 4 of theN-acetylgalactosamine (GalNAc) sugar, is found in nasal and trachealcartilages of bovines and porcines. It is also found in the bones,flesh, blood, skin, umbilical cord, and urine of these animals.Chondroitin-6 sulfate, also carbon 6 of the GalNAc sugar, has beenisolated from the skin, umbilical cord, and cardiac valves of theseanimals. Chondroitin-6 sulfate has the same composition, but slightlydifferent physical properties from chondroitin-4 sulfate. Chondroitinsulfate is involved in the binding of collagen and is also directlyinvolved in the retention of moisture. These are both properties thataid the healing process. A person skilled in the art will appreciatethat the terms “chondroitin sulfate,” “CS,” “chondroitin,” “chondroitinsulfuric acid,” and “chonsurid” are used interchangeably herein and alsoencompass chemical or isomeric or cross-linked derivatives throughoutthis application.

Dermatan sulfate (DS), also called chondroitin sulfate B, is mainly madeup disulfated and/or trisulfated disaccharide units of L-iduronic acidand N-acetyl-D-galactosamine joined by β1,4 or 1,3 linkages, but thereis a case where some of the repeating units contain sulfated L-iduronicacid or D-glucuronic acid as uronic acid, or contain non-sulfatedN-acetylgalactosamine or 4,6-disulfated N-acetylgalactosamine instead ofN-acetylgalactosamine-4-sulfate. DS is defined as a chondroitin sulfateby the presence of GalNAc. The presence of iduronic acid (IdoA) in DSdistinguishes it from chondroitin sulfates-A (4-O-sulfated) and -C(6-0-sulfated) and likens it to heparin and HS, which also contain thisresidue. It is considered that dermatan sulfate is absorbed by the bodywhen orally taken. The molecular weight and concentration of DS canvary, as discussed above with respect to the GAG component, but in anexemplary embodiment the molecular weight is in the range of about 10 to80 kDa and a concentration in the range of about 0.1-100 mg/ml. A personskilled in the art will appreciate that, unlike HA which is bacteriallyfermented and therefore has a molecular weight that can be controlled,dermatan sulfate is isolated from animal tissue and may containfragments. The molecular weight of the dermatan sulfate, and anyfragments contained therein, can therefore significantly vary. A personskilled in the art will also appreciate that the terms “dermatansulfate,” “DS,” and “dermatan” are used interchangeably herein and alsoinclude sulfated derivatives of dermatan sulfate, the dermatan sulfatebenzethonium salt, the persulfated derivatives of dermatan sulfatebenzethonium salts, and also the dermatan sulfate sodium salt.

Heparin and heparan sulfate (HS) are composed of a glucuronic acid(GlcA) linked to N-acetylglucosamine. They are composed of α1-4 linkeddisaccharide repeating units containing a uronic acid and an aminosugar. Heparan sulfate proteoglycans are an integral part of thebasement membrane. HA is a large biomolecule with a molecular mass asgreat as 400 kDa, composed of a core protein covalently bound to heparansulfate chains. The number of the polysaccharide chains and the size ofthe core protein may vary according to the source. Heparan sulfateproteoglycan is a multifunctional molecule binding to fibroblast growthfactors, vascular endothelial growth factor (VEGF), and VEGF receptorsthrough the sugar moiety, acting as a docking molecule for matrilysin(MMP-7) and other matrix metalloproteinases and playing important rolesin cell proliferation and differentiation. Heparan sulfate proteoglycansalso promote attachment of cells by binding to a variety of moleculesfound in the extracellular matrix including laminin, fibronectin,collagen type IV, and FGF-basic. The molecular weight and concentrationof HS can vary, as discussed above with respect to the GAG component,but in an exemplary embodiment the molecular weight is in the range ofabout 3-30 kDa (when isolated from tissues) and a concentration in therange of about 0.1-100 mg/ml.

Keratan sulfate, also keratosulfate (KS), is highly-negatively chargedand found principally in aggrecan, the most abundant proteoglycan in theextracellular matrix of hyaline, fibrous and elastic cartilage. KS iscomposed of disaccharide repeating unit, −4GlcNAcβ1-3Galβ1−. Sulfationoccurs at carbon position 6 (C6) of either or both the galactose (Gal)or GlcNAc. Specific KS types are composed of three regions: alinkageregion, at one end of which the KS chain is linked to the core protein,the repeating disaccharide unit, and chain capping region, occurring atthe opposite end of the KS chain to the protein linkage region. Themolecular weight and concentration of KS can vary, as discussed abovewith respect to the GAG component, but in an exemplary embodiment themolecular weight is in the range of about 5-10 kDa (when isolated fromtissues) and a concentration in the range of about 1-100 mg/ml.

A person skilled in the art will appreciate that, while lyophilized GAGsare particularly preferred, liquid GAGs can also be used to at leasttemporarily reduce the viscosity of HA. For example, CS can be obtainedin powder form and mixed with a solvent, such as water, to form asolution. The solution can be combined with HA to form a mixture havinga reduced viscosity, as compared to the HA alone. While effective toreduce the viscosity of the HA, the resulting mixture will have areduced concentration due to the presence of water. Thus, while liquid,non-lyophilized GAGs can be used with the present invention, in anexemplary embodiment the GAG is lyophilized to allow for the use of HAat a high concentration.

Lyophilization

Any one or more of the components present in the compositions andmethods of the present invention can be lyophilized using varioustechniques known in the art. Lyophilization is a dehydration processthat is typically used to preserve a perishable material, and it worksby freezing the material and then reducing the surrounding pressure andadding enough heat to allow the frozen water in the material to sublimedirectly from the solid phase to the gas phase. Standard lyophilizationtechniques known in the art can be used to lyophilize any one or more ofthe components. In an exemplary embodiment, at a minimum the secondcomponent, namely one or more GAGs, is lyophilized.

Prior to lyophilization, various solvents can be used to form an aqueousmixture containing the component(s) to be lyophilized. In an exemplaryembodiment, the aqueous mixture is prepared by combining water with oneor more of the components. The component(s) can be present within themixture at various amounts, for example in the range of about 1 to 100mg/ml, and more preferably at about 50 mg/ml. In an exemplaryembodiment, the composition is sterilized using a filter, such as a 0.2μm filter, prior to lyophilization. One filtration technique which canbe used is aseptic filtration.

In one embodiment, the component(s) can be lyophilized using thefollowing cycle:

Freezing: from ambient temperature to 5° C. in 15 minutes

-   -   Hold at 5° C. for 100 minutes    -   Down to −45° C. in 50 minutes    -   Hold at −45° C. for 180 minutes

Primary Drying: set pressure at 50 mTorr

-   -   Shelf Up to −15° C. in 175 minutes    -   Hold at −15° C. for 2300 minutes

Secondary Drying: set pressure at 75 mTorr

-   -   Shelf Up to 25° C. in 200 minutes    -   Hold for 900 minutes

Cycle end: backfill with nitrogen to ˜730 Torr

-   -   Capping and crimping

The addition of a buffering agent can provide for improved solubilityand stability of the GAG in lyophilized formulations. Biocompatiblebuffering agents known in the art can be used, such as glycine; sodium,potassium, or calcium salts of acetate; sodium, potassium, or calciumsalts of citrate; sodium, potassium, or calcium salts of lactate; sodiumor potassium salts of phosphate, including mono-basic phosphate,di-basic phosphate, tri-basic phosphate and mixtures thereof. Thebuffering agents can additionally have glycine added to the compositionto function as a bulking agent.

Other Joint Supplements

The components and methods of the present invention can also includeadditional components or joint supplements to enhance the treatmenteffect. For example, glucosamine (C₆H₁₃NO₅) (“GlcN”) can enhancesynthesis of key components of synovial fluid by feeding both reactionsnecessary for the production of hyaluronan as well as for proteoglycans.GlcN is an amino sugar carrying four hydroxyl groups and an amine group,and it is a prominent precursor in the biochemical synthesis ofglycosylated proteins and lipids. GlcN is a naturally occurring moleculethat has nutritive and effector functions. For example, GlcN iscompatible with and promotes stem cell growth and differentiation ofmesenchymal stem cells to form chondrocytes. GlcN can have a role intissue development and repair, such as cartilage growth and development,in general. In one embodiment, the GlcN can by lyophilized together withthe CS. Salt forms of glucosamine can have limited stability in liquidphase. In addition, HCl salt of GlcN can lower the pH sufficiently todegrade the HA once combined. For this reason, in order to retainstability of the components, it is advantageous to have GlcN in thelyophilized form to be solubilized with HA gel prior to injection.

The concentration of the GlcN can vary. A suitable local concentrationcan be at least about 10 mg/ml, about 9 mg/ml, about 8 mg/ml, about 7mg/ml, about 6 mg/ml, 5 mg/ml, 4.5 mg/ml, 4 mg/ml, 3.5 mg/ml, 3 mg/ml,about 2.9 mg/ml, about 2.8 mg/ml, about 2.7 mg/ml, about 2.6 mg/ml,about 2.5 mg/ml, about 2.4 mg/ml, about 2.3 mg/ml, about 2.2 mg/ml,about 2.1 mg/ml, about 2.0 mg/ml, about 1.9 mg/ml, about 1.8 mg/ml,about 1.7 mg/ml, about 1.6 mg/ml, about 1.5 mg/ml, about 1.4 mg/ml,about 1.3 mg/ml, about 1.2 mg/ml, about 1.1 mg/ml, about 1.0 mg/ml,about 0.9 mg/ml, about 0.8 mg/ml, about 0.7 mg/ml, about 0.6 mg/ml,about 0.5 mg/ml or so on. A person skilled in the art can determine asuitable local concentration of GlcN practicing methods known in thepharmaceutics art, and that determination will govern the nature andcomposition of the GlcN composition of interest to obtain the desiredconcentration of GlcN.

A person skilled in the art will appreciate that the compositions andmethods of the present invention can include various other jointtreatment or excipients, including, for example, amino acids, proteins,nucleic acids, buffers, surfactants and mixtures thereof. Steroids,anti-inflammatory agents, non-steroidal anti-inflammatory agents,analgesics, cells, stabilizers, antibiotics, antimicrobial agents,anti-inflammatory agents, growth factors, growth factor fragments,small-molecule wound healing stimulants, hormones, cytokines, peptides,antibodies, enzymes, isolated cells, platelets, immunosuppressants,nucleic acids, analgesics, cell types, viruses, virus particles, andcombinations thereof.

Composition

The first and second components, as well as any additional components,can be combined to form a composition that is able to be injected intotissue. As indicated above, one or more of the components, andpreferably at least the second component (the GAG), is lyophilized andis therefore a solid. In the event all components are lyophilized, thecomponents can be provided in one or more containers, and thenreconstituted with water, saline, buffer, or another solvent. Thesolubilization of a lyophilized or solid HA with a lyophilized GAG canallow for faster solubilization of HA. Where one or more of thecomponents is a liquid, and one or more of the components is a solid,the solid component(s) can be provided in a first container or in afirst chamber of a container, and the liquid component(s) can beprovided in a second container or in a second chamber of a container.The solid component(s) can be solubilized by the liquid component(s) orwith a diluent. Once the components are fully solubilized, thecomposition will at least temporarily exhibit a significantly reducedviscosity, as compared to the viscosity of HA alone, thus allowing thecomposition to be injected into tissue using a syringe.

In one embodiment, the second component is lyophilized and the firstcomponent is in an aqueous solution. The components are provided inseparate containers or separate chambers, and just prior to or duringsurgery the lyophilized second component is reconstituted with the firstcomponent. In particular, HA can be provided in liquid form, andlyophilized GAG can be combined with the HA. Once fully solubilized inthe HA, the viscosity of the HA will be significantly reduced, at leasttemporarily, allowing for mixing of the two components and ease ofinjection of the mixture. Alternatively, the lyophilized secondcomponent can be constituted with water, saline, or other solvent, andthen combined with the HA to form an injectable mixture.

In another embodiment, the first and second components are bothlyophilized and provided in solid form. The components can be providedin separate containers or chambers, or they can be provided in a singlecontainer or chamber. The components can be reconstituted with water,saline, or other solvent to form an injectable mixture. The lyophilizedsecond component, e.g., one or more GAGs, will have the affect ofreducing the viscosity of the mixture, at least temporarily.

In an exemplary embodiment, the components are configured to be combinedintraoperatively, i.e., immediately before or during an operation. Thecomponents, when combined, can form a resulting ready-to-use compositionor mixture having each component present in the composition at variousamounts. The amount of each component in the ready-to-use compositioncan vary, but in an exemplary embodiment the second component, e.g., oneor more GAGs, is present at an amount that is sufficient to reduce theviscosity of the first component, e.g., HA. For example, the firstcomponent, e.g., HA, and the second component, e.g., GAG, can be mixedat a ratio of the first component to the second component in the rangeof about 1:0.001 to 1:200, and in some embodiment of about 1:0.005 to1:100. The resulting concentration of the first component in theready-to-use composition can thus be in the range of about 0.6% to 4%%or more by weight of the total composition and the resultingconcentration of the second component in the ready-to-use compositioncan thus be in the range of about 0.001% to 10%. More preferably, thefirst component can be present in the ready-to-use composition at aconcentration of about 0.6%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or more byweight of the total composition, and the second component can be presentin the ready-to-use composition at a concentration of about 0.001%,0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more by weightof the total composition. In an exemplary embodiment, the amount of HApresent in the disclosed compositions is about 1.5-2.5% by weight of thetotal composition, and the amount of GAG present in the disclosedcompositions is about 0.001 to 10% by weight of the total composition.

Other solvents that can be used to solubilize one or more of thecomponents include, for example, saline or other salt solutions, buffersolutions such as phosphate buffered saline, histidine, lactate,succinate, glycine, and glutamate, dextrose, glycerol, water, as well ascombinations thereof. The compositions can also include othercomponents, such as dispersion media, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Isotonic agents include, for example, sugarssuch as dextrose, polyalcohols such as mannitol, sorbitol, or salts suchas sodium chloride in the composition. The composition can also includeminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the composition.

The components and/or the resulting composition can be sterilized priorto use using various techniques known in the art. Sterile injectablemixtures can be prepared by incorporating the active compound(s) in atherapeutically effective or beneficial amount in an appropriate solventwith one or a combination of ingredients, as required, followed byaseptic filtrations to yield a sterile formulation. Generally,dispersions are prepared by incorporating a compound(s), such as HA,into a sterile vehicle which contains a basic dispersion medium and anyrequired other ingredients. In the case of sterile powders for thepreparation of sterile injectable mixtures, some methods can includepreparation of vacuum dried and freeze-dried components which yield apowder of the composition plus any additional desired ingredients from apreviously sterile-filtered mixture thereof.

Method of Use

In use, the first and second components can be combinedintraoperatively, and then delivered to the treatment site, e.g., viainjection. In one embodiment, the first and second components can becombined, as discussed above, to form a mixture or composition.Combination of the lyophilized second component, e.g., one or more GAGs,with the first component, e.g., HA, can induce a transient reduction inviscosity of the HA mixture, at least temporarily, allowing greater easeof manipulating and allowing the combined HA/GAG mixture to be placedinto a syringe and injected into a patient. In certain embodiments, itmay be necessary to wait an amount of time for complete solubilizationof the lyophilized component(s) prior to injecting the composition intotissue. The amount of time it takes for the lyophilized second componentto solubilized in the first component, or within a solvent, can varydepending on the particular second component used, as well as themolecular weights and concentrations of the components. In general, itcan take anywhere from 10 seconds to 30 minutes for the second componentto be fully solubilized within the first component, or to bereconstituted with a solvent.

Once the lyophilized component is fully solubilized and the viscosity ofthe first component, e.g., HA, is significantly reduced, the viscositywill remain reduced for an amount of time, which again can varydepending on the particular materials used, as well as the molecularweight and concentration of the components within the composition. Incertain embodiment, once the second component is fully solubilized, theamount of time within which it is desirable to inject the compositioninto tissue can range from about 1-60 minutes, and more preferably from1-30 minutes, and more preferably the amount of time can be any one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60 minutes or greater or any variable herein.

Delivery Systems

As indicated above, the components used in the present compositions andmethods are preferably configured to be combined intraoperatively. Thecomponents can thus be provided as part of a kit. In one embodiment, thekit can include a first component, such as HA, and a second component,such as at least one GAG. The kit can also include additionalcomponents, as discussed above. The components can be housed in a singlecontainer or chamber, or in separate containers or chambers, of ahousing, such as a vial or syringe.

In an exemplary embodiment, the components are provided in a singlemixing and delivery device. The benefits of a single delivery capabilityinclude increasing the speed and ease of drug administration; reducingrisk of hospital acquired infection by reducing the number ofconnections; lowering the risk of drug administration or sequenceerrors; and quicker delivery of compositions requiring combination priorto administration.

FIG. 1 illustrates one embodiment of a mixing and delivery system thatis in the form of a dual chamber syringe 10. As shown, the dual chambersyringe 10 generally include a housing having proximal and distalchambers 12, 14 separated by a valve 16. A plunger 18 is slidablydisposed within the proximal chamber 12 and is configured to injectfluid present within the proximal chamber 12 into the distal chamber 14to thereby mix the components. In one embodiment, the first component,e.g., liquid HA, can be present in the proximal chamber 12 and thesecond component, e.g., lyophilized GAG, can be present in the distalchamber 14. The plunger 18 can be advanced through the proximal chamber12 to inject the first component, e.g., liquid HA, into the distalchamber 14 containing the second component, e.g., one or morelyophilized GAGs. In another embodiment, the proximal chamber 12 cancontain a solvent, such as water or saline, and the distal chamber 14can contain all of the components in solid form. For example, the distalchamber 14 can contain lyophilized or solid HA and one or morelyophilized GAGs. The plunger 18 can be advanced through the proximalchamber 12 to inject the solvent into the distal chamber 14, therebysolubilizing the components in the distal chamber 14. Once allcomponents are combined in the distal chamber 14, and any solid is fullysolubilized, the mixture can be delivered to tissue, for example byattaching a needle to the distal end of the dual chamber syringe.

FIG. 2 illustrates another embodiment of a mixing and delivery system20, which is sold commercially under the trade name MixJect®. In thisembodiment, the system includes a fluid control assembly 22 that iscoupled between a syringe 24 and a vial 26. The syringe 24 defines afirst chamber 24 a which can contain a liquid, such as liquid HA or asolvent, and the vial defines a second chamber 26 a which can contain asolid, such as one or more lyophilized GAGs. Deployment of the plunger28 through the syringe 24 will inject the liquid through the controlsystem and into the vial 26, where the solid will be solubilized by theliquid. Once the components are fully solubilized, the vial 26 can beinverted and the plunger 28 can be retracted to draw the mixture backinto the chamber 24 a in the syringe 24. The vial 26 can then be removedfrom the system, and the mixture can be injected from the syringethrough a needle 29 and into tissue.

A person skilled in the art will appreciate that any dual chambersystems known in the art can be used, and that the chambers can beside-by-side chambers with separate syringe plungers that mix into asingle chamber or linear chambers with a single plunger.

Prefilled syringes can contain the exact deliverable dose of desiredcompounds and diluents. By way of non-limiting example, the prefilledsyringes can contain volumes from about 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml,0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.5 ml, 2 ml, 2.5 ml, 3ml, 3.5 ml, 4 ml, 4.5 ml, 5 ml, 5.5 ml, 6 ml, 6.5 ml, 7 ml, 7.5 ml, 8ml, 8.5 ml, 9 ml, 9.5 ml, 10 ml or more, or any derivative thereof.

Treatment

The HA as well as the GAGs useful in the method and compositions can beadministered, for in vivo application, parenterally by injection or bygradual perfusion over time independently or together. Administrationmay be intravenously, intraperitoneally, intramuscularly,subcutaneously, intra-articular, intracavity, or transdermally. Examplesof symptoms or diseases, in which the composition and methods can exertactivity in treating articular disorders, include arthritis caused byinfections, injuries, allergies, metabolic disorders, etc., rheumatoidssuch as chronic rheumatoid arthritis, and systemic lupus erythematosus;articular disorders accompanied by gout, arthropathy such asosteoarthritis, internal derangement, hydrarthrosis, stiff neck,lumbago, etc. Varying the effects depending on the use of thecomposition or the types of diseases to be treated, the agent can exertdesired prophylactic and alleviative effects, or even therapeuticeffects on swelling, pain, inflammation, and destroying of articulationswithout seriously affecting living bodies. The composition for treatingarticular disorder can be used to prevent the onset of articulationdisorders, as well as to improve, alleviate, and cure the symptoms aftertheir onsets.

The methods of treatment can include directly injecting the compositionsinto the target area, such as a joint. Injections can be performed asoften as daily, weekly, several times a week, bi-monthly, several timesa month, monthly, or as often as needed as to provide relief ofsymptoms. For intra-articular use, from about 0.1 to about 500 mg perjoint, depending on the size of the joint and severity of the condition,can be injected. The frequency of subsequent injections into a givenjoint are spaced to the time of recurrence of symptoms in the joint.Illustratively, dosage levels in humans of the composition can be: knee,about 0.1 to about 500 mg per joint injection; shoulder, about 0.1 toabout 500 mg per joint injection; metacarpal or proximalintraphalangeal, about 0.1 to about 100 mg. per joint injection; andelbow, about 0.1 to about 300 mg per joint injection.

It will be understood, however, that the specific dosage level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

In one embodiment, the medical condition is osteoarthritis (OA) and thecomposition is administered in a joint space, such as, for example, aknee, shoulder, temporo-mandibular and carpo-metacarpal joints, elbow,hip, wrist, ankle, and lumbar zygapophysial (facet) joints in the spine.The viscosupplementation may be accomplished via a single or multipleintraarticular injections administered over a period of weeks into theknee or other afflicted joints. For example, a human subject with kneeOA may receive one, two, or three injections of about 2, 3, 4, 5, 6, 7,8, 9, 10 ml or more per knee. For other joints, the administered volumecan be adjusted based on the size on the joint.

In another exemplary embodiment, a kit is provided having first andsecond components. The first component can comprise 2 ml of Orthovisc®,which contains 30 mg of hyaluronan, 18 mg of sodium chloride, and up to2.0 mL of water for injection. The HA has a molecular weight in therange of about 1.0 to 2.9 MDa. The second component can comprise 0.1-100mg/ml of lyophilized bovine derived CS, supplied by, for example,Bioiberica of Barcelona, Spain and lyophilized using the protocoldiscussed above. The two components can be combined, e.g., using theaforementioned delivery systems, to solubilize the lyophilized CS. Fullsolubilization can take about X minutes. Once fully solubilized, themixture will have a significantly reduced viscosity, allowing themixture to be injected intra-articularly, e.g., into the knee joint.Injection preferably occurs within 5-30 minutes of solubilization.Additional injections can be once a week for a total of three of fourinjections. Standard intra-articular injection site preparation, aseptictechnique and precautions should be used.

Experimental Data

EXAMPLE 1 Lyophilization

A mixture containing 50 mg/ml glucosamine HCl and 50 mg/ml chondroitinsulfate was prepared and filtered with a 0.2 μm filter. The mixture wasthen filled in 3-mL glass vials at 1 mL/vial and lyophilized. Thelyophilized cake appeared to be solid and white. Each lyophilized samplewas able to be reconstituted with water, Orthovisc, or other HA gels,for injection. These HA gels were at 10-20 mg/ml concentrations and theHA was sourced from Shiseido and Lifecore. Samples were also able to bereconstituted with other buffers such as PBS.

EXAMPLE 2 Viscosity

Various amounts of different GAGs were lyophilized, as described above,to form a fluffy white cake. The GAGs include bovine derived CS,supplied by Bioiberica of Barcelona, Spain, heparin, supplied bySigma-Aldrich of Saint Louis, MO, and dextran sulfate, supplied bySigma-Aldrich of Saint Louis, Mo. The lyophilized GAGs were each placedin a 5 ml vial having an outer diameter of about 20.8 mm and a heightabout 40 mm. 40 mg of non-lyophilized GAGs was also placed in a 5 mlvial having the same dimensions. 2 ml of Orthovisc®, manufactured byAnika Therapeutics of Bedford, MA was injected into each vial and the HAwas allowed to fully dissolve the GAG. A control containing only 2 ml ofOrthovisc® was placed in a vial having the same dimensions.

The amount of time it took to fully solubilize the lyophilized GAG wasmeasured for some of the compositions and the results are set forthbelow. The viscosity of the mixture is measured by determining the “flowrate,” which is used herein to refer to the amount of time it took forthe contents of each vial to flow from one end of the vial to the otherend of the vial when the vial is turned upside-down. The results are setforth in the Table 1 below. The amount of time it took for the flow rateto increase to 1 minute was also measured and is set forth in the Table1 below. All results set forth in Table 1 are based on the average ofthree measurements.

TABLE 1 Time to fully Flow Rate of Time for Flow Rate to solubilizeComposition Increase to a Flow Lyophilized GAG After Fully Rate of 1Minute after Composition with HA Solubilized fully solubilized Control:not measured 20 minutes not applicable 2 ml HA (Orthovisc ®) 4 mg notmeasured 18 seconds 2 minutes lyophilized CS + 2 ml HA (Orthovisc ®) 40mg 5 minutes 9 seconds 15 minutes lyophilized CS + 2 ml HA (Orthovisc ®)60 mg not measured 4 seconds >30 minutes lyophilized CS + 2 ml HA(Orthovisc ®) 80 mg 15 minutes 5 seconds >30 minutes lyophilized CS + 2ml HA (Orthovisc ®) 50 mg <1 minute 7 seconds 5 minutes lyophilizedHeparin + 2 ml HA (Orthovisc ®) 50 mg <1 minute 12 seconds 5 minuteslyophilized Dextran Sulfate + 2 ml HA (Orthovisc ® ) 40 mg non- 15-20minutes 2 minutes Not applicable lyophilized CS powder + 2 ml HA(Orthovisc ®)

As shown in Table 1, HA alone is extremely viscous, with a flow rate ofabout 20 minutes for 2 ml of HA to transfer from one end of a 5 ml, 40mm high vial to the opposite end. When combined with GAG powder asreceived from the vendor or non-lyophilized GAG, there were localizedareas of low viscosity. However, during the time it takes to fullysolubilize all the powder, the mixture reverts back to high viscosity.Therefore, there is no time to fully mix the sample and get it back intothe syringe of a Mixject system. When combined with a lyophilized GAG,on the other hand, the HA is able to dissolve the GAG relatively quicklyand the viscosity was significantly reduced, at least temporarily, suchthat the mixture was able to transfer from one end of a vial to theopposite end of the vial in seconds. In most cases, with the exceptionof heparin and dextran, the viscosity remained at a significantlyreduced level for at least about 15 minutes.

EXAMPLE 3 Viscosity

40 mg of non-lyophilized CS powder, obtained from Bioiberica ofBarcelona, Spain, was mixed with 500 μL of water. The solution was thencombined with 2 ml of Orthovisc®, manufactured by Anika Therapeutics ofBedford, Mass.. After about 1-2 minutes of gentle mixing, the componentswere fully mixed. The flow rate was measured to be 45 seconds.

500 μL of water was combined with 2 ml of Orthovisc®, manufactured byAnika Therapeutics of Bedford, Mass. After about 3 minutes of gentlemixing, the flow rate was measured to be 26 seconds.

The combination of liquid CS with HA showed a reduced flow rate, ascompared to the combination of water and HA. Accordingly, the experimentdemonstrates the affects of CS on the viscosity of HA.

EXAMPLE 4 Solubilization

40 mg of non-lyophilized CS powder was obtained from Bioiberica ofBarcelona, Spain, was mixed with 2 ml of Orthovisc®, manufactured byAnika Therapeutics of Bedford, Mass. After about 15-20 minutes of gentlemixing, the CS was fully solubilized in the HA. Localized areas ofdecreased viscosity were observed during solubilization, however priorto full solubilization the HA gel reverted back to its high viscositystate, preventing transfer of the contents into another syringe forinjection.

Similarly, 40 mg of lyophilized CS powder, obtained from Bioiberica ofBarcelona, Spain, was mixed with 2 ml of Orthovisc®, manufactured byAnika Therapeutics of Bedford, Mass. After about 5 minutes of gentlemixing, the lyophilized CS was fully solubilized in the HA, and theviscosity of the HA was visibly decreased. The reduced viscosityremained for a sufficient period of time, allowing the mixture to bedrawn into a syringe for injection.

Terminology

A “therapeutically effective amount” or “effective amount” is thatamount of a agent to achieve a pharmacological effect. The term“therapeutically effective amount” includes, for example, aprophylactically effective amount. An “effective amount” is an amounteffective to achieve a desired pharmacologic effect or therapeuticimprovement without undue adverse side effects. For example, aneffective amount refers to an amount that increases operativity, orincreases the ability of the patient to bear load on the joint, ordecreases pain, or allows patient to get back to normal activity, orstimulate repair in the bone and cartilage of one or more joints, orreduces joint distortion, pain, swelling, inflammation, or stiffness.The effective amount of an agent will be selected by those skilled inthe art depending on the particular patient and the disease level. It isunderstood that “an effective amount” or “a therapeutically effectiveamount” can vary from subject to subject, due to variation in metabolismof therapeutic agents such as s and/or prokinetic agents, age, weight,general condition of the subject, the condition being treated, theseverity of the condition being treated, and the judgment of theprescribing physician.

“Treat” or “treatment” refers to any treatment of a disorder or diseaseassociated with bone or cartilage disorder, such as preventing thedisorder or disease from occurring in a subject which may be predisposedto the disorder or disease, but has not yet been diagnosed as having thedisorder or disease; inhibiting the disorder or disease, e.g., arrestingthe development or progression of the disorder or disease, relieving thedisorder or disease, causing regression of the disorder or disease,relieving a condition caused by the disease or disorder, or stopping thesymptoms of the disease or disorder. Thus, as used herein, the term“treat” is used synonymously with the term “prevent.”

By “co-administered” is meant simultaneous administration in the sameformulation or in two different formulations that are combined into oneformulation for administration. In one embodiment, the HA and the GAGare co-administered via delivery in the same formulation.

The term “subject” as used herein refers to an animal, preferably amammal and more preferably humans, horses, and dogs who can benefit fromthe compositions and methods of the present invention. There is nolimitation on the type of animal that could benefit from the presentmethods. A subject regardless of whether a human or non-human animal maybe referred to as an individual, subject, animal, host or recipient. Themethods of the present invention have applications in human medicine,veterinary medicine as well as in general, domestic or wild animalhusbandry.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

What is claimed is:
 1. A composition effective for injection in a jointof a patient, comprising: a mixture of a hyalauronic acid and aglycosaminoglycan, wherein the mixture has a first viscosity suitablefor injection through a syringe within a first period of time afterformation of the mixture and a second viscosity that is greater than thefirst viscosity following the first period of time.
 2. The compositionof claim 1, wherein the glycosaminoglycan comprises chondroitinsulphate.
 3. The composition of claim 1, further comprising a thirdcomponent comprising glucosamine.
 4. The composition of claim 1, whereinthe glycosaminoglycan is lyophilized.
 5. The composition of claim 1,wherein the hyaluronic acid has a molecular weight of at least of atleast about 1.0 MDa.
 6. The composition of claim 1, wherein thehyaluronic acid has a molecular weight of between about 1.0 MDa and 5.0MDa.
 7. The composition of claim 1, wherein the first period of time isat least 2 minutes.
 8. The composition of claim 1, wherein the firstperiod of time is at least 5 minutes.
 9. The composition of claim 1,wherein the first period of time is at least 15 minutes.
 10. Thecomposition of claim 1, wherein the solvent is selected from groupconsisting of water, saline, and buffer.
 11. The composition of claim 1,wherein the hyaluronic acid has a concentration of at least about 7mg/mL in the composition.
 12. The composition of claim 1, wherein thehyaluronic acid has a concentration between about 7 mg/mL and about 60mg/mL in the composition.
 13. The composition of claim 1, wherein theglycosaminoglycan has a concentration of at least about 2 mg/mL.
 14. Thecomposition of claim 1, wherein the glycosaminoglycan has aconcentration of at least about 5 mg/mL.